DNA-based vaccination of fish

ABSTRACT

The present invention relates to methods of immunization of aquaculture species by introducing DNA expression systems into the aquaculture species. Such DNA expression systems preferably include DNA sequences encoding polypeptides of pathogens of species of aquaculture. The present invention also relates to methods of administration of DNA expression systems into aquaculture. Such methods include injection, spray, and immersion techniques. The methods of this invention are useful for prophylactic vaccination or therapeutic immunization of fin-fish, shellfish, or other aquatic animals against infectious diseases.

Benefit of the United States Provisional Application No. 60/006,290,filed Nov. 7, 1995 is claimed herefor.

BACKGROUND OF THE INVENTION

Viral and bacterial diseases in fin-fish, shellfish or other aquaticlifeforms pose a serious problem for the aquaculture industry. Owing tothe high density of animals in the hatchery tanks or enclosed marinefarming areas, infectious diseases may eradicate a large proportion ofthe stock in, for example, a fin-fish, shellfish, or other aquaticlifeforms facility. Prevention of disease is a more desired remedy tothese threats to fish than intervention once the disease is in progress.Vaccination of fish is the only preventative method which may offerlong-term protection through immunity.

The fish immune system has many features similar to the mammalian immunesystem, such as the presence of B cells, T cells, lymphokines,complement, and immunoglobulins. Fish have lymphocyte subclasses withroles that appear similar in many respects to those of the B and T cellsof mammals. Additionally, the efficiency of the immune response of fishcan be affected by outside stresses, as is true in mammals. However,fish, unlike mammals, display a temperature-dependent development ofprotective immunity in response to antigens.

Most vaccines for fish have been developed against bacteria while therehave been very few fish vaccines made for combating viral or parasiticdiseases. Fish have been immunized by antigen-based immunization methodsusing live attenuated pathogens, killed whole pathogens, or morerecently, in laboratory settings, recombinant proteins. While liveattenuated vaccines induce good humoral and cell-mediated immuneresponses and can be administered orally or by immersion or injection,there is the important risk of reversion to a virulent form. Whole liveattenuated vaccines are not preferred in industrial farming due to therisk of contaminating other fish--a live attenuated vaccine which may begenerally safe for the target species of fish may be virulent in otherspecies of fish.

Fish vaccines using whole killed bacteria (i.e. bacterins) orrecombinant proteins from pathogens expressed in cell lines (subunitvaccines) have the disadvantage of inducing short-lived immuneresponses. Injected antigen, including recombinant protein, is processedsolely in an exogenous form usually causing induction of a humoralresponse (i.e., production of antibodies) but often a failure to inducecell-mediated immunity (i.e., cytotoxic T-cells).

Another disadvantage of whole killed and subunit vaccines is that theyalmost always must be injected and they require an adjuvant to induce aneffective immune response. Intramuscular injections of these adjuvantscan cause granuloma formation which scars the flesh and lowers themarket value of the fish. Intraperitoneal injection of adjuvants maycause adhesions between the viscera which can affect the health of thefish and retard fish growth.

Recombinant protein vaccines are difficult and expensive to makeespecially if the protein must be purified. For example,bacterially-expressed recombinant proteins may form inclusion bodiesfrom which recovery of protein in correct configuration may be low ornonexistent. Induction of an immune response may require that theantigenic protein be correctly glycosylated and folded, which may not beaccomplished in a cell other than an animal cell.

Some of the current methodologies for administering vaccines are nottechnically or economically practical. For example, direct injection ofrecombinant and whole killed pathogen vaccines into the fish is laborintensive and expensive relative to the future market value of the fish.Furthermore, injection needles can cross-infect fish with contaminatingpathogenic organisms, and accidental injection of humans can causesevere or fatal infections and anaphylactic reactions. Moreover,noninjurious injection of small fish is very difficult, especially inyoung fry, which are particularly susceptible to disease.

A less expensive and easier method which has been used to administerkilled viral or bacterial vaccines is an oral method wherein the vaccineis added directly to the water or incorporated into fish food. Oralvaccines have historically shown inconsistent and relatively low levelsof protection suggesting that they may be best used as a method ofrevaccination.

Genes have been introduced directly into animals by using live viralvectors containing particular sequences from an adenovirus, anadeno-associated virus, or a retrovirus genome. The viral sequencesallow the appropriate processing and packaging of a gene into a virion,which can be introduced to animals through invasive or non-invasiveinfection. Viral vectors have several disadvantages. Viral vectors beinglive pathogens, still carry the risk of inadvertent infection.Furthermore, proteins from viral vector sequences induce undesirableinflammatory or other immune responses which may prevent the possibilityof using the same vector for a subsequent vaccine or boost. Viralvectors also limit the size of the target gene that can be expressed dueto viral packaging constraints.

Naked DNA transfects relatively efficiently if injected into skeletalmuscle but poorly or not at all if injected into other tissues (Wolff etal., Science 247:1465-1468 (1990), incorporated herein by reference).Plasmid DNA coated onto the surface of small gold particles andintroduced into the skin by a helium-driven particle accelerator or"gene-gun" can directly transfect cells of the epidermis and dermis(Pecorino and Lo, Current Biol., 2:30-32 (1992), which is incorporatedherein by reference).

DNA has also been introduced into animal cells by liposome-mediated genetransfer. DNA-liposome complexes, usually containing a mixture ofcationic and neutral lipids, are injected into various tissues orinstilled into the respiratory passages. Nabel et al., Hum. Gene Ther.,3:649-656 (1992), which is incorporated herein by reference, have shownthat liposomes may be used to transfect a wide variety of cell types byintravenous injection in mammals. In addition, liposome-mediated genetransfer has been used to transfer the cystic fibrosis transmembraneconductance gene into the nasal epithelium of mice and humans sufferingfrom cystic fibrosis (Yoshimura et al., Nucleic Acids Reg., 12:3233-3240(1992) and Caplan et al., Nature Med., 1:39-46 (1995), respectively,both of which are incorporated herein by reference.

Substances may also be administered using biodegradable microspherescomposed of polymers such as polyester poly(lactide-co-glycolide) (Marxet al., Science, 260:1323-1328 (1993), incorporated herein byreference). It is notable that these particles can survive the upperdigestive system and arrive intact in cells of gut-associated lymphoidtissue (Eldridge et al., Adv. Exp. Med. Biol., 251:191-202 (1989),incorporated herein by reference). Biodegradable microspheres have beenused to deliver recombinant antigens, toxoids or attenuated virus intomammals by systemic and oral routes (O'Hagan et al., Immunology73:239-242 (1991); O'Hagen et al., Vaccine 11:149-154 (1993); Eldridgeet al., Mol. Immunol. 228:287-293 (1991) incorporated herein byreference). They may also be useful to deliver recombinant plasmid DNAto gut-associated lymphoid tissue for the purpose of immunization.

While most work has been carried out on mammals, plasmid DNA encodingreporter genes have been successfully introduced into fish byintramuscular injection (Hansen et al., FEBS Lett. 290:73-76 (1991),incorporated herein by reference). Thus, cells in fish can expressproteins from a foreign gene with the same types of vector constructs(i.e., backbones, promoter and enhancer elements) that are used inmammals.

The induction of an immune response to a protein expressed from anintroduced gene was first suggested by Acsadi et al., New Biologist3:71-81 (1991), which is incorporated herein by reference, who foundthat after plasmid DNA transfer into rat cardiac muscle, reporter geneexpression was transient but could be prolonged by treatment with animmuno-suppressant. Subsequently, it was shown that antibodies wereinduced in rodents against human growth hormone (Tang et al., Nature,356:152-154 (1992); Eisenbraun et al., DNA Cell. Biol., 12:791-797(1993), both of which are incorporated herein by reference) or humanα-antitrypsin (Tang et al., Nature, 356:152-154 (1992), alsoincorporated herein by reference) when these proteins were expressedfrom DNA coated onto gold particles and introduced into cells of theskin by bombardment.

DNA-based immunization refers to the induction of an immune response toan antigen expressed in vivo from a gene introduced into the animal.This method offers two major advantages over classical vaccination inwhich some form of the antigen itself is administered. First, thesynthesis of antigen in a self-cell mimics in certain respects aninfection and thus induces a complete immune response but carriesabsolutely no risk of infection. Second, foreign gene expression maycontinue for a sufficient length of time to induce strong and sustainedimmune responses without boost.

Several mammalian animal models of DNA-based immunization againstspecific viral, bacterial or parasitic diseases have been reported.These include influenza (Fynan et al., Proc. Nat'l Acad. Sci. USA,90:11478-11482 (1993); Montgomery et al., DNA Cell. Biol., 12:777-783(1993); Robinson et al., Vaccine, 11:957-960(1993); Ulmer et al.,Science, 259:1745-1749 (1993)!, HIV Wang et al. (1993)!, hepatitis BDavis et al., Hum. Molec. Genet., 2:1847-1851 (1993)!, malaria Sedagahet al., Proc. Nat'l Acad. Sci. USA, 91:9866-9870 (1994)!, bovine herpesCox et al., J. Virol, 67:5664-5667 (1993)!, herpes simplex Rousse etal., J. Virol., 68:5685-5689 (1994); Manicken et al. J. Immunol.,155:259-265 (1995)!, rabies Xiang et al., Virology, 199:132-140 (1994)!;lymphocytic choriomeningitis Yokoyama et al., J. Virol., 6964:2684-2688(1995)! and tuberculosis Lowrie et al., Vaccine, 12:1537-1540 (1994)!,all of which are incorporated herein by reference. In most of thesestudies a full-range of immune responses including antibodies, cytotoxicT lymphocytes (CTL), T-cell help and (where evaluation was possible)protection against challenge was obtained. In these studies naked DNAwas introduced by intramuscular or intradermal injection with a needleand syringe or by instillation in the nasal passages, or the naked DNAwas coated onto gold particles which were introduced by a particleaccelerator into the skin.

There is a need for novel systems to vaccinate fin-fish, shellfish, andother aquatic animals against diseases. These systems should beinexpensive to produce and administer, avoid the use of live, attenuatedorganisms, and induce strong and long-lasting immunity preferablywithout boost and with induction of both antibodies and cell-mediatedimmunity. More preferably, the system should be applicable to smallfish, be less stressful to fish during administration, and have thecapacity of simultaneously immunizing many animals for reducedlabor-related costs.

SUMMARY OF THE INVENTION

The present invention relates to the immunization of cultured fin-fish,shellfish, or other aquatic animals ("aquaculture species") by DNAexpression systems to overcome many disadvantages associated withantigen-based vaccines. The present invention relates to introduction ofDNA plasmids (alone or in a formulation) containing sequences encodingantigenic components of viral, bacterial or parasitic diseases bytransfection into aquaculture species. The methods and compositions ofthis invention are useful for immunization (i.e., for prophylacticvaccination or therapeutic immunization) of fin-fish, shellfish or otheraquatic animals against infectious diseases. The DNA sequences accordingto this invention are preferably present in vectors capable of inducingprotein expression of these sequences (i.e. expression vectors) and maybe administered alone or in combination with other DNA sequences in thesame or other expression vectors or as oligonucleotides. Theseadditional DNA sequences may encode cytokines, costimulatory molecules,or may include immunostimulatory sequences (e.g., CpG motifs). The DNAsequences may also be given with other adjuvants, such as alum.

The present invention also relates to methods of administration of DNAexpression vectors to aquaculture species, which may or may not encodepolypeptides from pathogens. DNA vectors of this invention may beadministered to aquaculture species by oral route, injection, spray, orimmersion. In a preferred embodiment, the DNA expression vectors of thisinvention are administered by immersion techniques or automatedinjection devices.

DESCRIPTION OF THE INVENTION

The present invention provides for methods and compositions forimmunizing cultured fin-fish, shellfish, and other aquatic animalsagainst infection by viral, bacterial or parasitic pathogens. In basicoutline, DNA encoding a polypeptide component of a pathogen isintroduced into an animal, and the polypeptide is expressed in cells ofthe animal, thus inducing an immune response that confers protectionagainst natural infection by the pathogen or helps overcome an ongoingand possibly chronic infection.

In a preferred embodiment, the present invention provides a method forimmunizing cultured fin-fish, shellfish, or other aquatic animalsagainst disease, comprising immersion of the animals in an aqueoussolution containing formulated plasmid DNA encoding one or moreantigenic determinants of an infectious agent (regardless of codonusage), whereby the DNA enters cells of the animal where it is expressedleading to induction of immune responses. The immunization procedure maybe prophylactic to prevent infection from occurring or may betherapeutic to treat pre-existing infections.

Few anti-viral vaccines have been marketed for fish. This is largely dueto the difficulty of growing virus in culture for the production ofwhole killed viral vaccines or safe attenuated strains of virus.Antigen-based vaccines using purified recombinant proteins are difficultand expensive to produce in large scale and may have poor immunogenicityin fish.

DNA-based immunization has several advantages. The antigenic protein issynthesized in vivo giving rise to both humoral and cell-mediated(cytotoxic T lymphocytes) immune responses. However, unlike liveattenuated pathogens, which also synthesize protein in vivo, DNAvaccines carry no risk of inadvertent infection. Unlike antigen-basedimmunization, DNA-based vaccination does not require the use oftraditional adjuvants to generate an effective immune response.Furthermore, DNA used in the methods of this invention is inexpensiveand easy to manufacture and purify.

DNA-based immunization also allows the host animal to produce foreignantigens within its own tissue thereby resulting in several advantages.One advantage is the efficient presentation of the foreign antigen tothe immune system due to the expression of a protein within a self-cell,which could be an antigen-presenting cell. Another advantage is thecorrect folding, protein modification, and disulfide bonding of aprotein expressed in a host cell, especially for viral proteins, whichare normally produced in cells of hosts. Recombinant viral proteinssynthesized in bacterial or yeast cells may be incorrectlypost-translationally modified and are often massed in inclusion bodies,which make the proteins difficult to purify or ineffective ifadministered in unpurified form.

Immune responses in fish are temperature dependent. Antigen-basedvaccines may give rise to sub-optimal immune responses if such vaccinesare given at the wrong temperature. DNA-based immunization isadvantageous because expression of the antigenic protein could continueover a long period until such time as to stimulate an immune responsewhen the temperature is optimal.

Another advantage of prolonged synthesis of antigen is the induction ofimmune responses as soon as the immune system is mature. Fish may beunable to induce sufficient immune responses at a young age. Forexample, trout and halibut may not produce lymphoid cells until as lateas ten and thirty days after hatching, respectively, and T-dependentimmune responses do not appear until months after hatching. Using themethods of this invention, expression of foreign protein in fish cancontinue at least four months after transfection indicating thatDNA-based immunization may be preferred for vaccination of young fish.

The term "vaccine" herein refers to a material capable of producing animmune response. A vaccine according to this invention would produceimmunity against disease in cultured fin-fish, shellfish and otheraquatic species. One of skill in the art would readily appreciate thatactivation of CTL activity resulting from in vivo synthesis of antigenwould produce immunity against disease not only prophylactically butalso therapeutically (after development of disease in culture).

Aquaculture species treated by methods of this invention will include adiversity of species of cultured fin-fish, shellfish, and other aquaticanimals. Fin-fish include all vertebrate fish, which may be bony orcartilaginous fish. A preferred embodiment of this invention is theimmunization of fin-fish. These fin-fish include but are not limited tosalmonids, carp, catfish, yellowtail, seabream, and seabass. Salmonidsare a family of fin-fish which include trout (including rainbow trout),salmon, and Arctic char. Examples of shellfish include, but are notlimited to, clams, lobster, shrimp, crab, and oysters. Other culturedaquatic animals include, but are not limited to eels, squid, and octopi.

Purification of DNA on a large scale may be accomplished by anionexchange chromatography (for example, resins manufactured by Qiagen,U.S. FDA Drug Master File (DMF-6224)).

DNA which is introduced to aquaculture species will encode foreignpolypeptides (e.g., those derived from viral, bacterial or parasiticpathogens). Polypeptides of this invention refer to complete proteins orfragments thereof, including peptides which are epitopes (e.g., a CTLepitope) associated with an infectious virus, bacterium or parasite.

DNA sequences encoding a complete or large parts of an antigenic proteinare preferred where humoral immunity is desired rather than DNAsequences encoding smaller parts, such as only CTL epitopes, as arepreferred where cell-mediated immunity is desired and humoral immunitymay be deleterious. In preferred embodiments, the DNA sequences encodingpolypeptides of viral pathogens may be selected from the groupconsisting of glycoprotein (G) or nucleoprotein (N) of viral hemorrhagicsepticemia virus (VHSV); G or N proteins of infectious hematopoieticnecrosis virus (IHNV); VP1, VP2, VP3 or N structural proteins ofinfectious pancreatic necrosis virus (IPNV); G protein of spring viremiaof carp (SVC); and a membrane-associated protein, tegumin or capsidprotein or glycoprotein of channel catfish virus (CCV).

In other preferred embodiments, the DNA sequences encoding polypeptidesof bacterial pathogens may be selected from the group consisting of aniron-regulated outer membrane protein, (IROMP), an outer membraneprotein (OMP), and an A-protein of Aeromonis salmonicida which causesfurunculosis, p57 protein of Renibacterium salmoninarum which causesbacterial kidney disease (BKD), major surface associated antigen (msa),a surface expressed cytotoxin (mpr), a surface expressed hemolysin(ish), and a flagellar antigen of Yersiniosis; an extracellular protein(ECP), an iron-regulated outer membrane protein (IROMP), and astructural protein of Pasteurellosis; an OMP and a flagellar protein ofVibrosis anguillarum and V. ordalii; a flagellar protein, an OMPprotein, aroA, and purA of Edwardsiellosis ictaluri and E. tarda; andsurface antigen of Ichthyophthirius; and a structural and regulatoryprotein of Cytophaga columnari; and a structural and regulatory proteinof Rickettsia.

In yet another preferred embodiment, the DNA sequences encodingpolypeptides of a parasitic pathogen may be selected from one of thesurface antigens of Ichthyophthirius.

The methods of this invention could also be used to introduce plasmidvectors encoding polypeptides endogenous to the animal, but which mightbe normally present in low concentrations (e.g., growth hormones). Inthis case the expression proteins would serve a physiological role (i.e.enhanced growth) rather than induce an immune response.

Vectors useful in the making of expression plasmids include, but are notlimited to, vectors containing constitutive promoters, induciblepromoters, tissue-specific promoters, or promoters from the gene of theantigen being expressed. Constitutive promoters may include strong viralpromoters, for example, promoter sequences from cytomegalovirus (CMV),Rous sarcoma virus (RSV), simian virus-40 (SV40), or herpes simplexvirus (HSV). Tissue-specific promoters may include the muscle beta-actinpromoter or the thymidine kinase promoter. An inducible or regulatablepromoter, for example, may include a growth hormone regulatablepromoter, a promoter under the control of lac operon sequences or anantibiotic inducible promoter or a Zinc-inducible metallothioneinpromoter.

The vector should include an expression control sequence comprising apromoter (e.g., inducible or constitutive promoters described above) DNAsequence, and may include, but is not limited to, an enhancer element,an RNA processing sequence such as an intronic sequence for splicing ofa transcript or a polyadenylation signal (e.g., from simian virus-40(SV40) or bovine growth hormone (BGH)), a signal sequence for secretionof the expressed protein, or one or more copies of immunostimulatory DNAsequences known as CpG motifs. The vector should also include one ormore of the following DNA sequences: bacterial origin of replicationsequences, a selectable marker, which may be for antibiotic resistance(e.g., kanamycin) or for non-antibiotic resistance (e.g.,β-galactosidase gene).

Oligonucleotides having unmethylated CpG dinucleotides have been shownto activate the immune system (A. Krieg, et al., "CpG motifs inBacterial DNA Trigger Directed B Cell Activation" Nature 374:546-549(1995)). Depending on the flanking sequences, certain CpG motifs may bemore immunostimulatory for B cell or T cell responses, andpreferentially stimulate certain species. Copies of CpG motifs in DNAexpression vectors act as adjuvants facilitating the induction of animmune response against an expressed protein. A CpG motif, a stretch ofDNA containing CpG dinucleotides within a specified sequence, may be asshort as 5-40 base pairs in length. Multiple CpG motifs may be insertedinto the non-coding region of the expression vector. When a humoralresponse is desired, preferred CpG motifs will be those thatpreferentially stimulate a B cell response. When cell-mediated immunityis desired, preferred CpG motifs will be those that stimulate secretionof cytokines known to facilitate a CD8+ T cell response.

Other CpG motifs have be found to inhibit immune responses. In apreferred embodiment of the application, these immunoinhibitory CpGmotifs would be removed or mutated in a DNA expression vector used bythe methods of this invention, without disrupting the expression ofpolypeptides therefrom.

An additional preferred embodiment of this invention relates to theadministration of a vector containing one or more different DNAsequences, one sequence encoding an antigen and the others encodingpolypeptides which may or may not be antigenic. For example, the vectormay encode two antigens from the same pathogen. Alternatively, thedifferent antigen(s) may induce an immune response against a differentpathogen and thus serve as a multivalent vaccine. Alternatively, theother polypeptides may serve to enhance an immune response against atargeted pathogen (e.g., helper epitopes, cytokines, carrierpolypeptides, cholera toxin subunits, or other immunostimulants).

When two or more polypeptide-encoding DNA sequences are present in onevector, the transcription of each antigen-encoding DNA sequence may bedirected from its own promoter. Alternatively, one promoter may drivethe expression of two or more antigen-encoding DNA sequences joined inframe to each other to express a fusion protein. For example, VP2 andVP3 proteins of infectious pancreatic necrosis virus (IPNV) may befused. In another embodiment, DNA sequences encoding two or moreantigens from different diseases may be joined to form a multivalentvaccine when expressed.

Alternatively, a DNA sequence encoding an antigenic polypeptide may befused to a DNA sequence encoding a carrier polypeptide. In a preferredembodiment, the carrier polypeptide may contain one or more envelopeproteins of the hepatitis B virus, preferably from the human hepatitis Bvirus. In a more preferred embodiment, the envelope proteins ofhepatitis B virus will be the small and major protein (also referred toas surface antigen).

In another embodiment, each polypeptide-encoding DNA sequence in thevector may be under the control of its own promoter for expression oftwo or more non-fused polypeptides.

Alternatively, the DNA sequences encoding additional antigens may beadministered by using a second vector containing such sequences. Suchsequences may encode antigens from the same pathogen or differentpathogens, or cytokines, cholera toxin subunits, or otherimmunostimulants. Such a vector may be administered concurrently orsequentially with the first expression vector. A preferred embodiment ofthis invention is the concurrent administration of expression vectors.One vector may be induced to express protein simultaneously with orafter expression of protein from the other vector.

In yet another embodiment of this invention, antigen-expressing vectorsmay be administered concurrently with an antigen-based vaccine such as arecombinant protein or whole-killed vaccine. In a preferred embodiment,the antigen-expressing vector is administered simultaneously with aprotein antigen (i.e. recombinant protein or whole killed pathogen).Another preferred embodiment would be to first administer a DNA vaccineto prime the immune response followed by administration of the proteinantigen two to eight weeks later, preferably orally or by immersion, toboost the immune response.

The DNA used in the method of this invention is preferably purifiedplasmid DNA(s) simply dissolved in an aqueous solution or in aformulation. One of skill in the art would readily appreciate how toformulate DNA used in the methods of this invention with knowntransfection reagents such as cationic liposomes, fluorocarbonemulsions, cochleates, tubules, gold particles, biodegradablemicrospheres, or cationic polymers.

Liposomes useful for transfection of DNA of this invention includecommercially available liposomes and liposomes containing eithercationic lipids or cationic polymers. In a preferred embodiment of thisinvention, liposomes would include a mixture of a neutral lipid such asdioleoylphosphatidylethanolamine (DOPE) or cholesterol and a cationiclipid.

In a more preferred aspect of the invention, liposomes would include amixture of cationic polymers and neutral lipids such as DOPE orcholesterol. Such liposomes may be prepared as described herein and inUnited States Provisional Patent Application entitled, "A Novel Class ofCationic Reagents for High Efficient and Cell-Type-Specific Introductionof Nucleic Acids into Eukaryotic Cells", incorporated by referenceherein. Unlike cationic lipids, cationic polymers do not haveester-linkages and have greater stability in vivo as a result. Cationicpolymers (also referred to as dendrimers) may be dimeric, cyclic,oligomeric, or polymeric in structure.

Cationic polymers in an aqueous solution without neutral lipids are alsopreferred transfection reagents according to the preferred embodimentsof this invention. Cationic polymers have been shown to work well fortransfecting fish cells in vitro with plasmids expressing fish pathogenantigens (see Table 1, Example 1).

Cochleates, which are stable phospholipid-calcium precipitates composedof phosphatidylserine, cholesterol and calcium are desirable non-toxicand non-inflammatory transfection reagents that can survive thedigestive system. Biodegradable microspheres composed of polymers suchas polyester poly(lactide-co-glycolide) have been used tomicroencapsulate DNA for transfection.

Tubules have been previously described in the literature as lipid-basedmicrocylinders consisting of helically wrapped bilayers of lipid, theedges of which are packed together. DNA may be placed in the hollowcenter for delivery and controlled release in animals.

With immersion, DNA may enter cells of the epithelium of the skin, thegills or the gut wall. With injection, DNA may enter muscle cells orother cells in muscle tissue (e.g. fibroblasts, immune cells) or cellsof viscera within the intraperitoneal cavity. DNA may then be expressedin these transfected cells leading to induction of appropriate immuneresponses in regional or systemic lymphoid tissue.

The invention provides for pharmaceutical compositions comprising DNAvaccines in an amount effective for the treatment and prevention ofdiseases caused by pathogens of aquaculture species. According toanother embodiment, the pharmaceutical compositions of this inventionfurther comprise a second DNA vaccine, an adjuvant, a recombinantprotein, a transfection reagent, or some combination thereof.

Methods of this invention may be useful in the immunization ofaquaculture species against many pathogens. Such pathogens include butare not limited to hemmorrhagic septicemia virus, infectioushematopoietic necrosis virus, infectious pancreatic necrosis virus,virus causing spring viremia of carp, channel catfish virus (Herpesvirusictaluri), grass carp hemorrhagic virus, nodaviridae such as nervousnecrosis virus or striped jack nervous necrosis virus, infectious salmonanaemia virus, Aeromonis salmonicida, Renibacterium salmoninarum,Yersinia, Pasteurella (including piscicida), Vibrosis (includinganguillarum and ordalii), Edwardsiella (including ictaluri and tarda),Streptococci, and Ichthyophthirius.

In one embodiment of this invention, recombinant plasmid DNA isintroduced into animals orally. DNA for oral use may be formulated withbiodegradable microspheres, fluorocarbon emulsions, cochleates, ortubules. This is a non-stressful method of immunizing aquaculturespecies by which DNA may be coated onto or milled into feed in the formof a paste or liquid suspension or incorporated into gelatin capsulesand introduced into the environment of the aquaculture species.Preparations of DNA for oral use may include lactose and corn starch.The DNA can be used with or without products to enhance entry into cellsof the gut epithelium or more deeply situated cells.

In another embodiment, pure recombinant plasmid DNA is introduced intoanimals by injection with a needle or a jet-injection system, which doesnot have a needle. Injection areas of the fin-fish include but are notlimited to intraperitoneal, intramuscular, and subcutaneous areas of thefish. In a preferred embodiment, large fin-fish are immunized byinjection methods of this invention. Typically, fish are injected with0.1-0.5 ml of a solution containing DNA. DNA may be injected in a pureform or may be formulated with liposomes, cationic polymers,fluorocarbon emulsions, cochleates, or tubules.

In yet another embodiment of this invention, pure DNA is introduced intoa fin-fish by particle bombardment. This method introduces DNA-coatedgold particles into the epidermis of a fin-fish using a "gene-gun",which uses compressed helium to shoot the gold particles at high speedinto the skin. This method has been shown to be particularly efficientfor induction of cell-mediated immune responses with small quantities ofDNA in mice.

In another embodiment of this invention, plasmid DNA is introduced tofish by spray. Typically, fish are exposed to spray for at least 2seconds. Fish may pass through a mist of DNA solution by forcing thevaccine through high-pressure paint-sprayer-type nozzles. Typically, anypressure up to 90 psi is satisfactory. Due to the number of pounds offish per unit volume that can be vaccinated by spray, it may be moreeconomical to immunize larger fish by this method than by immersion. TheDNA can be used with or without products to enhance entry into cells ofthe skin. For example, the DNA may be associated with liposomes orcationic polymers.

In a more preferred embodiment of this invention, a large number ofanimals can be immunized simultaneously by immersion in a solutioncontaining DNA. In one embodiment, fish are dip-netted into suspensionscontaining DNA formulations (e.g., DNA formulated with cationic polymersor liposomes) for at least several seconds. The fish are then returnedto the holding tanks in which they develop immunity. In anotherembodiment, fin-fish, shellfish, or other aquatic animals are placedinto tanks containing a relatively small volume of water. ConcentratedDNA formulations (e.g., DNA formulated with cationic polymers orliposomes) is added to the tank, and animals are left for a period oftime up to several hours before the tank is refilled with water torestore the normal aquatic environment. This method of immersion ispreferred for the immunization of small fry, which cannot be immunizedby direct injection.

The amount of the expression plasmid DNA that may be combined with acarrier material to produce a single dosage form will vary dependingupon the host treated, and the particular mode of administration. Itshould be understood, however, that a specific dosage and treatmentregimen for any particular fish will depend upon a variety of factors,including the expression of the particular plasmid DNA employed, thestability and activity of the particular protein or peptide expressed,age, body weight, general health, species of fish, the progress of thedisease being treated, and nature of the disease being immunized againstor dreaded. The amount of expression plasmid DNA may also depend uponwhether other therapeutic or prophylactic agents including additionalexpression plasmid DNAs and adjuvants, if any, are co-administered withthe expression plasmid.

Without being bound by the values listed below, dose ranges for theadministration of DNA used in the methods of this invention may begeneralized as follows. For immunization of fish via oral routes, 0.1 to50 μg DNA per fish administered over several consecutive days may beused. For DNA-based immunization by intramuscular or intraperitonealinjection, 0.1 to 10 μg of DNA may be used. For spray immunization, avolume of 1 ml per fish of 0.1 to 10 mg/ml DNA solution may be useful.Fish immunized by immersion methods of this invention may be incubatedin a 1 to 100 μg/ml DNA solution at a volume sufficient for fish tosurvive for a time period necessary for uptake of DNA to produce animmune response by the fish. An effective dosage range for immunizationof fish via gene-gun route may be 10 ng to 1 μg.

Adjuvants for immunization are well known in the art and suitableadjuvants can be combined with the DNA sequences described herein by aperson skilled in the art to form a pharmaceutical composition. Oiladjuvants are least desirable for the methods of this invention becausethey create undesirable side-effects such as visceral adhesions (whichcan restrict growth) and melanized granuloma formations (which can lowerthe grade of the fish at market) and because they cannot form ahomogeneous mixture with DNA preparations. DNA-based immunization doesnot require oil adjuvants and thus avoids these undesirable effects.

Adjuvants used in immunization with DNA expression plasmids of thisinvention may include alum or a DNA molecule having unmethylated CpGdinucleotides therein (also referred to as CpG adjuvant).Oligonucleotides having unmethylated CpG dinucleotides have been shownto activate the immune system (A. Krieg, et al., "CpG motifs inBacterial DNA Trigger Directed B Cell Activation" Nature 374:546-549(1995)). CpG motifs may be inserted into a plasmid DNA vaccine vector,and replicated in bacteria thereby allowing the CpG motifs to retaintheir unmethylated form. As such, administration of a CpG adjuvantcloned into plasmid vectors would be simultaneous with theadministration of a plasmid DNA vaccine. Alternatively, a CpG adjuvantin the form of free oligonucleotides may be administered before, duringor after the administration of a plasmid DNA vaccine.

Oligonucleotides having CpG motifs may be optionally modified at theirphosphodiester linkages for stability purposes. Such modifications arewell known by those of skill in the art. For example, phosphodiesterbonds in an oligonucleotide may be replaced by phosphorothioatelinkages.

The present invention also includes pharmaceutical products for all ofthe uses contemplated in the methods described herein. For example, apharmaceutical product comprising pure plasmid DNA vector orformulations thereof, operatively coding for an immunogenic polypeptideor peptide, may be prepared in physiologically acceptable administrableform (e.g., saline). The pharmaceutical product may be placed in acontainer, with a notice associated with the container in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the DNA for veterinary administration. Suchnotice, for example, may be labeling approved by the Biologics Divisionof Agriculture and Agri-Food Canada or the United States Department ofAgriculture (USDA) or the approved product insert.

In order that this invention may be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in anyway.

EXAMPLES Example 1 Cloning of DNA encoding Antigenic Proteins IntoPlasmid DNA Vectors

DNA encoding proteins of fish pathogens is useful in developing DNA fishvaccines. Table 1 below recites fish pathogen protein expressionplasmids. Table 1 describes nucleotide sequences encoding proteins frompathogens cloned into vectors having the cytomegalovirus promoter (CMV),i.e., pcDNA3 (from Invitrogen) or a vector containing the CMV promoterand intron A of CMV to promote better expression of protein (pCMV_(A)vector). For example, genetic sequences coding for the majorglycoprotein (G) or nucleoprotein (N) of the viral hemorrhagicsepticemia virus (VHSV) were cloned into the EcoRI site of either thepcDNA3 or pCMV_(A) vector. Nucleotide sequences encoding the VP2 and VP3structural proteins of the infectious pancreatic necrosis virus (IPNV)were cloned into same vectors. The gene encoding the ferric siderophorereceptor (fstA) of Aeromonas salmonicida has also been cloned into expression vectors. The fstA protein is one of several possibleiron-regulated outer membrane proteins that could be expressed as anantigen from a DNA vaccine.

                  TABLE 1                                                         ______________________________________                                        Plasmid Vector       Antigen     Pathogen                                     ______________________________________                                        pCMV-G  pcDNA3       G glycoprotein                                                                            viral hemorrhagic                                    (EcoRI site) (#1-1565)*  septicemia virus                             pCMV.sub.A -G                                                                         pCMV.sub.A vector                                                                          G glycoprotein                                                                            viral hemorrhagic                                    (EcoRI site) (#1-1565)*  septicemia virus                             pCMV-N  pcDNA3       N nucleoprotein                                                                           viral hemorrhagic                                    (EcoRI site) (#92-1306)* septicemia virus                             pCMV.sub.A -N                                                                         pCMV.sub.A vector                                                                          N nucleoprotein                                                                           viral hemorrhagic                                    (EcoRI site) (#92-1306)* septicemia virus                             pCMV-VP2                                                                              pcDNA3       VP2         infectious pan-                                                               creatic                                              (HindIII/XbaI site)                                                                        (#117-1760)*                                                                              necrosis virus                               pCMV.sub.A -                                                                          pCMV.sub.A vector                                                                          VP2         infectious pan-                              VP2                  creatic                                                          (Sall/XbaI)  (#117-1760)*                                                                              necrosis virus                               pCMV-VP3                                                                              pcDNA3       VP3         infectious pan-                                                               creatic                                              (EcoRI/XbaI site)                                                                          (#2325-3011)*                                                                             necrosis virus                               pCMV.sub.A -                                                                          pCMV.sub.A vector                                                                          VP3         infectious pan-                              VP3                              creatic                                              (EcoRI/XbaI site)                                                                          (#2325-3011)*                                                                             necrosis virus                               pCMV-fstA                                                                             pcDNA3       IROMP fstA  Aeromonis                                                                     salmonicida                                          (EcoRI/XbaI site)                                                                          (#76-2630)*                                              pCMV.sub.A -                                                                          pCMV.sub.A vector                                                                          IROMP fstA  Aeromonis                                    fstA                             salmonicida                                          (EcoRI/XbaI slte)                                                                          (#76-2630)*                                              ______________________________________                                         *# indicates the nucleotide sequences within the genome of the pathogen       which have been cloned to code for the antigen                           

Example 2 Expression of Foreian Protein In Fish Injected with PurePlasmid DNA Vector

The pCMV-luc plasmid used in the following experiments contains theluciferase reporter gene (luc) under the control of the cytomegaloviruspromoter. Purified plasmid DNA was prepared by using commerciallyavailable Qiagen DNA purification columns. The purified plasmid DNA wasthen dissolved in endotoxin-free Dulbecco's phosphate buffered saline(DPBS) without calcium chloride or magnesium chloride, or in 0.15M NaCldissolved in deionized distilled water for a final concentration of0.001 mg/ml to 5 mg/ml DNA. Fish were anaesthetized with 0.168 mg/mltricaine (3-amino benzoic acid ethylester) in water or by placing thefish on ice for 30-60 seconds before injection. Trout and zebra fishwere injected intramuscularly between the dorsal fin and the lateralline with 10 μl of the DNA solution.

Luciferase activity was measured in the muscle and gills 2.5 days afterinjection. Rainbow trout were euthanized by an overdose of tricaine(0.1% w/v). Zebra fish were killed by immersion in ice. The muscle orgills of the fish were removed on ice, homogenized, centrifuged topellet cellular debris, and the supernatants containing soluble proteinswere assayed for luciferase activity. Luciferase assays were carried outusing a kit commercially available from Promega Corporation. Lightemission in relative light units (RLU) was quantitated by a luminometer(Analytical Luminescence Laboratory) over a ten second interval andbackground values from control samples were subtracted. Theconcentration of protein in the supernatants was determined andluciferase activity was expressed as RLU/sec/mg protein.

The results summarized in Table 2 (below) indicate that purified plasmidDNA can efficiently transfect fish cells after intramuscular injection.Nanogram amounts of plasmid DNA were able to induce detectable proteinexpression in both the injected muscle as well as more distant cells(e.g., in gills), showing that different types of cells, possiblyincluding antigen presenting cells (APC), are transfectable by plasmidDNA and are capable of synthesis of foreign protein. Cells distant tothe site of injection (i.e. gills) expressed lower amounts of proteinthan the injected muscle cells. Zebra fish and trout are not closelyrelated species of fish. Therefore, the results in Table 2 indicate thatmost species of fish could take up and express foreign proteins frominjected plasmids.

                  TABLE 2                                                         ______________________________________                                        Dose     Luciferase Activity (RLU/sec/mg of protein)*                         of DNA   Trout               Zebra Fish                                       (μg)  Muscle    Gills       Muscle  Gills                                  ______________________________________                                        0.01     3,449     4         502     18                                                (±1548)                                                                              (±3)   (±307)                                                                             (±8)                                  0.1      22,768    36        11,665  94                                                (±12,708)                                                                            (±14)  (±2,989)                                                                           (±66)                                 1        78,408    618       826,486 228                                               (±51,523)                                                                            (±567) (±368,790)                                                                         (±115)                                10       280,051   982       199,285 833                                               (±172,749)                                                                           (±743) (±97,134)                                                                          (±621)                                50       417,226   980       145,891 5,519                                             (±165,164)                                                                           (±393) (±85,645)                                                                          (±4791)                               ______________________________________                                         *mean ± standard error of mean (n = 10 fish per group)                

Example 3 Kinetics And Longevity of Foreign Gene Expression in Fish

One microgram of pCMV-luc plasmid in 10 μl of saline was injectedintramuscularly into adult zebra fish and 3-4 month old rainbow trout aspreviously described in Example 2. Luciferase activity in the muscle andgills of the injected fish was determined at various times between 2.5days and 8 weeks using the methods described in Example 2. In Table 3(below), the plasmid DNA directs protein expression within days ofinjection and protein expression in post-mitotic muscle remains stablefor at least eight weeks. Luciferase expression in the gills falls offover time, possibly due to cell turnover.

                  TABLE 3                                                         ______________________________________                                               Luciferase Activity (RLU/sec/mg of Protein)*                           Time     Trout              Zebra Fish                                        (days)   Muscle   Gills       Muscle  Gills                                   ______________________________________                                        0        0        0         0       0                                         2.5      78,408   618       2,107,048                                                                             8,705                                              (±51,523)                                                                           (±567) (±1,281,284)                                                                       (±6853)                                14       54,004   211       4,160,080                                                                             6,965                                              (±19,411)                                                                           (±133) (±2,553,955)                                                                       (±3,672)                               28       90,686   39        5,236,613                                                                             6,056                                              (±46,044)                                                                           (±20)  (±4,536,744)                                                                       (±4048)                                56       18,219   30        6,395,781                                                                             3,246                                              (±11,785)                                                                           (±23)  (±1,764,195)                                                                       (±1,040)                               ______________________________________                                         *mean ± standard error of mean (n = 10 fish per group)                

Example 4 Kinetics and Longevity of Foreign Gene Expression in ZebraFish

Zebra fish were injected intramuscularly with 0.1 μg of purified plasmidpCMV-luc DNA in 10 μl of saline as previously described in Example 2.The results from Table 4 (below) indicate that even ten-fold less DNAthan used in Example 3 is capable of producing detectable levels ofprotein for at least sixteen weeks. The results also indicate thatprotein expression begins within hours after injection.

                  TABLE 4                                                         ______________________________________                                                         Luciferase Activity                                                           (RLU/sec/mg                                                  Time             of Protein)*                                                 (days)   Muscle          Gills                                                ______________________________________                                        0.16     64 (±35)     1 (±1)                                            0.33     1,620 (±1418)                                                                              3 (±2)                                            0.5      2,739 (±1359)                                                                              30 (±19)                                          1        2,629 (±1,129)                                                                             15 (±2)                                           2.5      11,665 (±2,989)                                                                            94 (±66)                                          112      82,424 (±49,208)                                                                           103 (±50)                                         ______________________________________                                         *mean ± standard error of mean (n = 10 fish per group)                

Example 5 Transfection of Fish Cells by Injection of Plasmid DNAFormulated With a Cationic Lipid

Zebra fish were injected intraperitoneally (IP) (i.e., in the abdomen)with 0.1 μg of pCMV-luc alone or associated with 0.5 μg of a cationiclipid, G304 (obtained from Gibco BRL, New York, USA) in 10 μl.

Luciferase activity in muscle, gills, and viscera (liver, spleen,intestine, stomach, swim bladder, pyloric caecae, and ovary or testis)was measured 2.5 days after DNA injection. The fish tissues wereprepared as described previously (Example 2).

Table 5 (below) shows that protein is expressed from DNA that isinjected intraperitoneally into fish. Injection of plasmid DNAformulated with cationic lipid resulted in higher foreign proteinexpression in the viscera than injection of DNA alone. Muscle tissue, onthe other hand, expressed greater levels of luciferase enzyme when theplasmid pCMV-luc DNA was injected without the cationic lipid. Therefore,a cationic lipid may increase transfection efficiency depending thetarget tissue.

                  TABLE 5                                                         ______________________________________                                                      Luciferase Activity (RLU/sec/mg                                 Zebra Fish    of protein)*                                                    Tissue    DNA alone     DNA + lipid                                           ______________________________________                                        Muscle    151 (±127) 32 (±10)                                           Gills     15 (±15)   8 (±8)                                             Viscera   3 (±2)     85 (±40)                                           ______________________________________                                         *mean ± standard error of mean (n = 10 fish per group)                

Example 6 Comparison of Foreign Gene Expression after Injection of DNAin Fish and in Mice

Fish and mice were injected intramuscularly with a range of 0.1 μg to50.0 μg of pCMV-luc plasmid DNA in 50 μl . Total luciferase activity forthe whole muscle of mouse or fish was assayed 2.5 days after injection.The fish and mouse tissues were prepared as described previously(Example 2).

For each dose of DNA, injected trout demonstrated higher levels ofluciferase activity than injected mice (Table 6 below). In general, fora given dose of DNA, luciferase activity was approximately 100 timeshigher in fish than in mouse. Therefore, the knowledge that mice can beimmunized against numerous diseases using doses of DNA within the rangetested here, and the finding that fish muscle is more easily transfectedand/or that fish muscle expresses transgenes more efficiently, indicatethat fish should be good candidates for DNA-based immunization.

                  TABLE 6                                                         ______________________________________                                                       Luciferase Activity (RLU/sec                                   Dose of DNA    total)*                                                        (μg)        mouse    trout                                                 ______________________________________                                        0.01           N/A      65,898                                                                        (±30,774)                                          0.1            1,649    327,724                                                              (±542)                                                                              (±177,583)                                         1.0            5,466    1,100,347                                                            (±1536)                                                                             (±669,634)                                         10             43,082   3,225,068                                                            (±5,419)                                                                            (±1,869,474)                                       50             70,713   4,520,741                                                            (±15,921)                                                                           (±1,609,457)                                       ______________________________________                                         *mean ± standard error of mean (n = 5 for trout except for the 50 μ     dose group for which n = 12; n = 10 for mouse groups)                    

Example 7 Expression of Plasmid DNA after Immersion of Fish inDNA-containing Solutions

Cationic lipid, G304, was obtained from Gibco BRL, New York, USA.Cationic polymer liposomes designated Q203, Q205, Q206, Q208, Q250, andQX were obtained from Qiagen GmbH, Hilden, Germany. Cationic polymerliposomes are composed of a mixture of cationic polymers and neutrallipids. Such transfection reagents were prepared as described in U.S.Provisional Patent Application entitled, "A Novel Class of CationicReagents for High Efficient and Cell-Type-Specific Introduction ofNucleic Acids into Eukaryotic Cells", incorporated by reference herein.

For example, a cationic polymer (either Q203, Q205, Q206, Q208, Q250 orQX, described below in Table 7) and a neutral lipid,dioleoyloxiphosphatidylethanolamine (DOPE) were mixed together for afinal concentration of 2 mM in chloroform, which was then evaporated offin a rotary evaporator at 60° C. The mixture was dried for 10 minutesunder a reduced pressure of 10 to 15 mbar. Under sterile conditions,endotoxin free deionized water was added to the mixture, which was thenheated while stirring at 60° C.

Next, Q203, Q205, Q250, and QX were sonicated once for 300 seconds at60° C. In the case of Q250, trans β carotene was added to a finalconcentration of 0.37 mM before sonication. Q206 and Q208 were notsonicated but were stirred at 60° C. until the solutions becametransparent or slightly opalescent. The total concentration ofDOPE+cationic polymer for all liposomes was 2 mM. The concentration ofDOPE in each liposome can be calculated by multiplying the X(DOPE) valuein Table 7 by 2 mM so that, for example, Q203-containing liposomes are1.7 mM DOPE and 0.3 mM Q203. Table 7 (below) summarizes the cationicpolymer liposomes used in the methods of this invention.

                  TABLE 7                                                         ______________________________________                                        Cationic                                                                      Polymer                                                                       Liposome                         Method of                                    Reagent                                                                              Cationic Polymers                                                                              X(DOPE)  Preparation                                  ______________________________________                                        Q203   butandiyl-1,4-bis(octadecyl                                                                             with sonication.                                    dimethylammonium bromide)                                                                      0.85                                                  Q205   butandiyl-1,4-bis(octadecyl                                                                    0.82     with sonication.                                    dimethylammonium bromide)                                              Q206   butandiyl-1,4-bis(octadecyl                                                                    0.78     without soni-                                       dimethylammonium bromide) cation.                                      Q208   butandiyl-1,4-bis(octadecyl                                                                    0.75     without soni-                                       dimethylammonium bromide) cation.                                      Q250   didodecyldimethyl ammonium                                                                     0.571    with sonication.                                    bromide                   Add trans β                                                              carotene to final                                                             concentration                                                                 of 0.37 mM.                                  QX     didodecyldimethyl ammonium                                                                     0.375    with sonication.                                    bromide                                                                ______________________________________                                    

DNA:liposome complexes were prepared by independently diluting DNA andliposome solutions in 0.15M NaCl, then mixing the two solutions andvortexing, and then incubating the mixture at room temperature for 30-45minutes. The solutions were diluted further with water and incubated foran additional 10-15 minutes at room temperature prior to use with fish.

Each fish was immersed in the solution of liposome formulated DNA (2.5ml or 5 ml per fish) for 90 minutes and then returned to its normalholding tank. After 2.5 days, the fish were homogenized or gills andmuscle were homogenized separately and assayed for luciferase activity.

Table 8 (below) shows luciferase activity above background in individualzebra fish after immersion. Thus, the results of Table 8 indicate thatthe majority of fish were successfully transfected and able to expressforeign protein after immersion in DNA:liposome solutions. No fishexpressed luciferase activity after immersion in pCMV-luc DNA withoutliposomes. Therefore, lipid-containing transfection reagents appear tosignificantly contribute to the transfection efficiency of DNA into fishwith the immersion technique.

                  TABLE 8                                                         ______________________________________                                        Transfection                                                                              Total Luciferase Activity                                         Reagent     (RLU/ second)                                                     ______________________________________                                        G304        330, 65, 0, 1643, 1581, 143, 5, 165, 0, 0, 257                    Q203        268, 82, 106, 264                                                 Q205        188, 268, 166, 136                                                Q206        208, 286, 170, 108, 174                                           Q208        668, 204, 1060, 0, 0, 180, 842, 242, 90, 36                       Q250        358, 398, 60, 10, 134, 1742, 54, 136, 84, 136                     QX          166, 80, 302, 74, 432, 630, 28, 28, 260, 260                      ______________________________________                                    

Example 8 Induction of an Immune Response against a Protein Derived froma Fish Pathogen in Fish and Mice by Injection of Antigen-EncodingPlasmid DNA

Purified pCMV_(A) -VP3 DNA (encoding the VP3 protein of infectiouspancreatic necrosis virus) was prepared as described previously forpCMV-luc DNA and injected intramuscularly in mice or trout. Each ofthree adult female BALB/C mice received 100 μg in tibialis anteriormuscle while a single one-year old female trout received 200 μg in thetail muscle. Two weeks later, the humoral immune response against theexpressed antigen was determined by ELISA assay of plasma taken from themice and fish to detect anti-VP3 antibody.

The ELISA assay was performed using standard techniques. In particular,96-well plates were coated with infectious pancreatic necrosis virus(IPNV) particles, blocked with a non-specific protein, and then washed.Ten-fold serial dilutions of trout or mice plasma and control plasma(obtained from non-injected mice and fish or from animals injected withcontrol DNA not encoding VP3) were put in the appropriate wells (100μl/well) were incubated for 2 hours. After washing, bound anti-VP3antibodies in trout and mouse plasma were detected by addition ofhorse-radish peroxidase-labeled (HRP) mouse anti-trout or goatanti-mouse IgG monoclonal antibodies, respectively. Amounts of boundantibody were quantitated by reaction with O-phenylenediaminedihydrochloride, which is cleaved by HRP producing a color measurable bya spectrophotometer at OD₄₅₀.

ELISA titer values in Table 9 (below) indicate the dilution factor whichgave an OD₄₅₀ value twice that of background. Table 9 shows thatDNA-based immunization of fish or mice by intramuscular injection ofplasmid DNA can induce an immune response against an antigenic proteinof a fish pathogen such as the VP3 protein of IPNV.

                  TABLE 9                                                         ______________________________________                                        Species    anti-VP3 ELISA titers                                              ______________________________________                                        Mouse      527.2                                                              Trout      14.1                                                               ______________________________________                                    

The collective results of the examples show the expression of foreignproteins in cells of fish after administration of pure plasmid DNA,either by intramuscular or intraperitoneal injection of pure orformulated plasmid DNA, or by injection of or immersion in DNAformulated with cationic liposomes. Furthermore, the collective resultsshow that an immune response can be induced if the protein is antigenic,for example a protein of a fish pathogen. This should lead to protectionagainst natural infection by virulent pathogen.

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that our basic construction can be altered toprovide other embodiments which utilize the methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the claims appended hereto rather than the specificembodiments which have been presented hereinbefore by way of example.

What is claimed is:
 1. A composition for inducing an immune response infinfish comprising:an expression vector having an expression controlsequence capable of directing expression in finfish of at least oneimmunogenic polypeptide and a polypeptide-encoding DNA sequence encodingat least one immunogenic polypeptide from a fish pathogen.
 2. Thecomposition according to claim 1, wherein the vector additionallycomprises an immunostimulatory unmethylated CpG motif.
 3. Thecomposition according to claim 1, wherein the injection of the vectorcomprising a fish pathogen induces a protective response in finfish. 4.The composition for inducing an response according to claim 1 or 3,wherein the vector is selected form the group consisting of pCMV-G,pCMV-N, pCMV-VP2, pCMV-VP3, pCMV-fstA, and.
 5. The composition accordingto claim 1, wherein the polypeptide-encoding DNA sequence additionallyencodes a different polypeptide from the same pathogen.
 6. Thecomposition according to claim 1, wherein the polypeptide-encoding DNAsequence additionally encodes a polypeptide from a different pathogen.7. The composition according to claim 1, wherein thepolypeptide-encoding DNA sequence additionally encodes a carrierpolypeptide to form a fusion protein with the immunogenic polypeptide.8. The composition according to claim 7, wherein the carrier polypeptideis a surface antigen of the human hepatitis B virus.
 9. The compositionaccording to claim 1, wherein the vector further comprises a secondexpression control sequence capable of directing expression of apolypeptide and a second polypeptide-encoding DNA sequence undertranscriptional control of the second expression control sequence. 10.The composition according to claim 9, wherein the secondpolypeptide-encoding DNA sequence is a cytokine.
 11. The composition forinducing an immune response according to claim 1 further comprising animmune modulator adjuvant.
 12. The composition according to claim 11,wherein the adjuvant is an oligonucleotide having an immunostimulatoryunmethylated CpG motif.
 13. The composition according to claim 1formulated with a transfection reagent.
 14. The composition according toclaim 13, wherein the transfection reagent is selected from the groupconsisting of liposomes, fluorocarbon emulsions, cochleates, tubules,gold particles, biodegradable microspheres, and cationic polymers. 15.The composition according to claim 14, wherein the transfection reagentis a liposome.
 16. The composition according to claim 14, wherein thetransfection agent is a cationic polymer.
 17. The composition accordingto claim 1, wherein the immunogenic polypeptide is encoded in the genomeof a rhabdovirus selected from the group of viral haemorrhagicsepticaemina virus (VHSV), infectious haematopoietic necrosis virus(IHNV), and spring viraemia of carp virus (SVCV).
 18. The compositionaccording to claim 1, wherein the immunogenic polypeptide is encoded inthe genome of a fish rhabdovirus selected from the group of viralhaemorrhagic septicaemina virus (VHSV), infectious haematopoieticnecrosis virus (IHNV), and spring viraemia of carp virus (SVCV).
 19. Thecomposition according to claim 1, wherein the immunogenic polypeptide isencoded in the genome of a birnavirus.
 20. The composition according toclaim 1, wherein the birnavirus is infectious pancreatic necrosis virus(IPNV).
 21. The composition according to claim 1, wherein theimmunogenic polypeptide is selected from the group consisting of VP1,VP2, VP3, and N structural proteins encoded in the genome of theinfectious pancreatic necrosis virus (IPNV).
 22. The compositionaccording to claim 1, wherein the immunogenic polypeptide is encoded bythe genome of a marine herpesvirus.
 23. The composition according toclaim 22, wherein the immunogenic polypeptide is encoded by the genomeof the channel catfish virus (CCV).
 24. The composition according toclaim 23, wherein the immunogenic polypeptide is selected from the groupconsisting of an envelope protein, a membrane-associated protein)tegumin, a capsid protein, and a glycoprotein of the channel catfishvirus (CCV).
 25. The composition according to claim 1, wherein theimmunogenic polypeptide is encoded by the genome of a marine nodavirus.26. The composition of claim 25, wherein the nodavirus is selected fromthe group consisting of nervous necrosis virus and striped jack nervousnecrosis virus.
 27. The composition according to claim 1, wherein theimmunogenic polypeptide is encoded by the genome of a iridovirus. 28.The composition according to claim 27, wherein the iridovirus isselected from the group consisting of fish lymphocystis disease virus(FLDV) and other marine iridoviruses.
 29. The composition according toclaim 1, wherein the immunogenic polypeptide is encoded by the genome ofinfectious salmon anaemia virus (ISAV).
 30. The composition according toclaim 1, wherein the immunogenic polypeptide is encoded by the genome ofa bacterial pathogen selected from the group consisting of Aeromonissalmonicida, Renibacterium salmoninarum, Vibrosis anguillarum andVibrosis ordalii, Yersiniosis, Pasteurellosis, Edwardsiellosis ictaluri,Edwardsiellosis tarda, Cytophaga colummari, and Rickettsia.
 31. Thecomposition according to claim 1, wherein the immunogenic polypeptide isselected from the group consisting of an iron-regulated outer membraneprotein, (IROMP), an outer membrane protein (OMP), and an A-protein ofAeromonis salmonicida.
 32. The composition according to claim 1, whereinthe immunogenic polypeptide is selected from the group consisting of thep57 protein, major surface associated antigen (msa), a surface expressedcytotoxin (mpr), and a surface expressed hemolysin (ish) ofRenibacterium salmoninarum.
 33. The composition according to claim 1,wherein the immunogenic polypeptide is a flagellar antigen ofYersinosis.
 34. The composition according to claim 1, wherein theimmunogenic polypeptide is selected from the group consisting of anextracellular protein (ECP), an iron-regulated outer membrane protein(IROMP), and a structural protein of Pasteurellosis.
 35. The compositionaccording to claim 1, wherein the immunogenic polypeptide is selectedfrom the group consisting of an outer membrane protein (OMP) and aflagellar protein of a member of the genus Vibrosis, wherein said memberis selected from the group consisting of Vibrosis anguillarum andVibrosis ordalii.
 36. The composition according to claim 1, wherein theimmunogenic polypeptide is selected from the group consisting of aflagellar protein, an outer membrane protein (OMP) protein, aroA, andpurA of a member of the genus Edwardsiellosis, wherein said member isselected from the group consisting of Edwardsiellosis ictaluri and E.tarda.
 37. The composition according to claim 1, wherein the immunogenicpolypeptide is a structural or regulatory protein of Cytophagacolumnari.
 38. The composition according to claim 1, wherein theimmunogenic polypeptide is a structural or regulatory protein ofRickettsia.
 39. The composition according to claim 1, wherein theimmunogenic polypeptide is encoded by the genome of a marine parasite.40. The composition according to claim 39, wherein the marine parasiteis a member of the genus Ichthyophthirius.
 41. The composition accordingto claim 39, wherein the immunogenic polypeptide is surface antigen ofthe parasite Ichthyophthirius.
 42. A method of inducing immune responsein fin-fish against infection from fish pathogens, comprisingadministering to the fin-fish a composition for inducing an immuneresponse comprising an expression vector having an expression controlsequence capable of directing expression of an immunogenic polypeptide,and a polypeptide-encoding DNA sequence encoding an immunogenicpolypeptide.
 43. The method according to claim 42, wherein the vectoradditionally comprises a nucleic acid sequence comprising at least oneunmethylated CpG motif, wherein the nucleic acid sequence isimmunostimulatory.
 44. The method according to claim 42, wherein thepolypeptide-encoding DNA sequence additionally encodes a differentpolypeptide from the same pathogen.
 45. The method according to claim42, wherein the polypeptide-encoding DNA sequence additionally encodes apolypeptide from a different pathogen.
 46. The method according to claim42, wherein the polypeptide-encoding DNA sequence additionally encodes acarrier polypeptide to form a fusion protein with the immunogenicpolypeptide.
 47. The method according to claim 42, wherein the vectorfurther comprises a second expression control sequence capable ofdirecting expression of a polypeptide and a second polypeptide-encodingDNA sequence under transcriptional control of the second expressioncontrol sequence.
 48. The method according to claim 42, wherein thecomposition is formulated with a transfection reagent selected from thegroup consisting of liposomes, fluorocarbon emulsions, cochleates,tubules, gold particles, biodegradable microspheres, and cationicpolymers.
 49. The method according to claim 42, further comprisingadministering to the fin-fish an additional DNA expression vectorcomprising an expression control sequence capable of directingexpression of an immunogenic polypeptide and a polypeptide-encoding DNAsequence capable of inducing an immune response.
 50. The methodaccording to claim 42, further comprising administering to the fin-fishan adjuvant before or after administration of the composition.
 51. Themethod according to claim 42, further comprising administering to thefin-fish a recombinant or purified protein, wherein said protein isadministered before, during or after administration of the composition.52. The method according to claim 42, wherein the composition isadministered by intramuscular injection or intraperitoneal injection.53. The method according to claim 42, wherein the composition isadministered by immersion.
 54. The method according to claim 53, whereinthe DNA is formulated with a transfection reagent selected from thegroup consisting of cationic liposomes and cationic polymers.
 55. Themethod according to claim 42, wherein the fin-fish is selected from thegroup consisting of salmon, salmonid, carp, catfish, trout (includingrainbow trout), yellowtail, seabream, and seabass.
 56. The methodaccording to claim 42, wherein the pathogen is a viral fish pathogen.57. The method according to claim 42, wherein said immunogenic peptideis encoded in the genome of a viral fish pathogen.
 58. The methodaccording to claim 42, wherein the immunogenic polypeptide is encoded inthe genome of a rhabdovirus selected from the group of viralhaemorrhagic septicaemina virus (VHSV), infectious haematopoieticnecrosis virus (IHNV), and spring viraemia of carp virus (SVCV).
 59. Themethod according to claim 42, wherein the immunogenic polypeptide isencoded in the genome of a birnavirus.
 60. The method according to claim59, wherein the birnavirus is an infectious pancreatic necrosis virus(IPNV).
 61. The method according to claim 42, wherein the immunogenicpolypeptide is selected from the group consisting of VP1, VP2, VP3, andN structural proteins encoded in the genome of the infectious pancreaticnecrosis virus (IPNV).
 62. The method according to claim 42, wherein theimmunogenic polypeptide is encoded by the genome of a marineherpesvirus.
 63. The method according to claim 62, wherein theimmunogenic polypeptide is encoded by the genome of the channel catfishvirus (CCV)
 1. 64. The method according to claim 63, wherein theimmunogenic polypeptide is an envelope protein of the channel catfishvirus (CCV).
 65. The method according to claim 42, wherein theimmunogenic polypeptide is encoded by the genome of a marine nodavirus.66. The method according to claim 65, wherein the nodavirus is selectedfrom the group consisting of nervous necrosis virus and striped jacknervous necrosis virus.
 67. The method according to claim 42, whereinthe immunogenic polypeptide is encoded by the genome of a iridovirus.68. The method according to claim 67, wherein the irridovirus isselected from the group consisting of fish lymphocystis disease virus(FLDV) and other marine iridoviruses.
 69. The method according to claim42, wherein the immunogenic polypeptide is encoded by the genome ofinfectious salmon anaemia virus (ISAV).
 70. The method according toclaim 42, wherein the immunogenic polypeptide is encoded by the genomeof a bacterial pathogen selected from the group consisting of Aeromonissalmonicida, Renibacterium salmoninarum, Vibrosis anguillarum andVibrosis ordalii, Yersiniosis, Pasteurellosis, Edwardsiellosis ictuluri,Edwardsiellosis tarda, Cytophaga columnari, and Rickettsia.
 71. Themethod according to claim 42, wherein the immunogenic polypeptide isselected from the group consisting of an iron-regulated outer membraneprotein, (IROMP), an outer membrane protein (OMP), and an A-protein ofAeromonis salmonicida.
 72. The method according to claim 42, wherein theimmunogenic polypeptide is selected from the group consisting of the p57protein, major surface associated antigen (msa), a surface expressedcytotoxin (mpr), and a surface expressed hemolysin (ish) ofRenibacterium salmoninarum.
 73. The method according to claim 42,wherein the immunogenic polypeptide is a flagellar antigen ofYersinosis.
 74. The method according to claim 42, wherein theimmunogenic polypeptide is selected from the group consisting of anextracellular protein (ECP), an iron-regulated outer membrane protein(IROMP), and a structural protein of Pasteurellosis.
 75. The methodaccording to claim 42, wherein the immumogenic polypeptide is selectedfrom the group consisting of an outer membrane protein (OMP) and aflagellar protein of a member of the genus Vibrosis, wherein said memberis selected from the group consisting of Vibrosis anguillarum and V.ordalii.
 76. The method according to claim 42, wherein the immunogenicpolypeptide is selected from the group consisting of a flagellarprotein, an outer membrane protein (OMP) protein, aroA, and purA of amember of the genus Edwardsiellosis, wherein said member is selectedfrom the group consisting of Edwardsiellosis ictaluri and E. tarda. 77.The method according to claim 42, wherein the immunogenic polypeptide isa structural or regulatory protein of Cytophaga columnari.
 78. Themethod according to claim 42, wherein the immunogenic polypeptide is astructural or regulatory protein of Rickettsia.
 79. The method accordingto claim 42, wherein the immunogenic polypeptide is encoded by thegenome of a marine parasite.
 80. The method according to claim 79,wherein the marine parasite is a member of the genus Ichthyophthirius.81. The method according to claim 42, wherein the immunogenicpolypeptide is a surface antigen of the parasite Ichthyophthiriusmultifiliis.
 82. A method for expressing a polypeptide in fin-fishcomprising administering to a fin-fish a DNA expression vectorcomprising an expression control sequence capable of directingexpression of a polypeptide and a polypeptide-encoding DNA sequenceencoding at least one polypeptide from a fish pathogen.
 83. The methodaccording to claim 82, wherein said DNA expression vector isadministered by a technique selected from the group consisting ofintramuscular injection, intraperitoneal injection, or immersion.